One of the most important human health-related scientific challenges is to understand how the immune system is able to protect against a wide variety of infectious agents. The broad objective of this grant is to investigate the biochemical basis of human immunodiversity. Two key events are required to produce high affinity antibodies from lower specificity antibodies, somatic hypermutation (SHM) and class switch recombination (CSR). Both antibody diversification events require the action of a recently discovered enzyme, activation induced cytidine deaminase (AID). Within the past year it has been shown that AID catalyzes deamination of cytosine to uracil on single-stranded DNA substrates. The deaminations observed in vitro occur most frequently in specific trinucleotide sequences corresponding to SHM hot spot sequences in vivo. Our specific goal, encompassed in four specific aims, is to understand the biochemical basis of SHM. We propose to address this problem first by characterizing the deamination specificity of purified AID in hotspot and coldspot sequences. We will carry out a kinetic analysis to determine binding and catalytic preferences for human wild type AID. We will then investigate AID mutants with altered C deamination specificity that no longer favor hotspot motifs. We will test the hypothesis that wild type AID properties are determined by its ability to scan DNA processively, in contrast to less processive AID mutants exhibiting reduced deamination specificity. Only actively transcribed immunoglobulin variable genes undergo SHM. In the third specific aim, we will investigate AID-catalyzed deamination using a eukaryotic transcription system, including RNA polymerase II and transcription factors. Using "classical" biochemical add-back experiments, we will perform a systematic search for transcription components that function to target AID to the transcription apparatus. The first three specific aims are intended to clarify the role of AID in initiating C to T transition mutations in the non-transcribed stand, at an important class of hotspot motifs in SHM. A longer-term goal, embodied in specific aim four, is to investigate mutational strand specificity, including mutations at A and T sequences. This aim will investigate AID in conjunction with base excision repair, mismatch repair and recently discovered error-prone DNA polymerases. [unreadable] [unreadable]